ON THE ROLE OF P53 IN THE CELLULAR RESPONSE TO ANEUPLOIDY by Akshay Narkar A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland July 2020 © 2020 Akshay Narkar All rights reserved ABSTRACT A majority of solid tumors are aneuploid and the tumor suppressor protein 53, has been implicated as the guardian of euploid genomes in animal organisms. Experiments using transformed or non-transform human cell lines showed that aneuploidy induction leads to p53 accumulation and a consequent, p21-mediated, G1 cell cycle arrest. However, it has been controversial as to whether there is a universal signal elicited by the aneuploid state that activates p53. My PhD thesis attempts to advance our understanding of the universality of the p53 response in different cell and tissue context post aneuploidy induction. In this study, we found that, whereas adherent 2-dimensional (2D) cultures of human immortalized or cancer cell lines indeed activate p53 upon aneuploidy induction as previously reported, suspension cultures of a human lymphoid cell line undergoes a p53- independent cell cycle arrest upon aneuploidy formation. To dive deeper into the molecular mechanisms regulating p53-independent arrest post aneuploidy in Nalm6 cells we performed a genome wide CRISPR/Cas9 knockout screen. This revealed previously uncharacterized functions of PDCD5 and LCMT1 in regulating chromosomal aberrations. Recent studies highlight that tissue environment plays a critical role in regulating chromosome segregation, however the role of p53 in 3D environment is not well characterized. To our surprise, 3-dimensional (3D) organotypic cultures of cells from human or mouse neural, intestinal or mammary epithelial tissues do not activate p53 or undergo G1 arrest upon aneuploidy induction. Instead, TP53-/- organoids exhibit increased ii aneuploidy production that correlates with mitotic aberrations and dysregulated cell cycle timing. Our data suggest that p53 is not necessarily a universal surveillance factor that prohibits the proliferation of aneuploid cells, but rather, it helps ensure stable chromosome transmission through a more direct role in mitotic fidelity. Primary Reader and Advisor: Rong Li Secondary Reader: Ben Ho Park iii ACKNOWLEDGEMENTS I am very grateful to my advisor, Professor Rong Li, for introducing me to the amazing world of cell biology, and for her guidance along the road of my PhD study. Rong’s profound scientific knowledge and broad vision in science have been a role model to me. I am also thankful to her for being patient and allowing me to explore different research ideas. Postulating multiple hypothesis and designing experiments to rigorously test it have not only helped me to shape my independent research but also made science enjoyable. I am grateful to Professor Roger Reeves, Ben Ho Park and Andrew Holland for always being supportive and serving on my thesis committee. Thanks to the Human Genetics program - Dr. David Valle, Dr. Kirby Smith, Dr. Haig Kazazian and Sandy Muscelli. I feel very proud to be a part of the Institute for Genetic Medicine at Hopkins. I also want to thank my classmates and colleagues from the department to make graduate life fun. A big thank you to my lab mates Linhao, Blake, Jin, Alexis, Soham, Wahid, Sree, Tiane and Albert for scientific and fun insights. Lastly, I am very grateful to my family for their infinite love and support, it would not have been possible without you!! iv Dedicated to my family, for their eternal support, love and trust. मा�ा कुटुंबासाठी, ां�ा शा�त समथ�नासाठी, प्रेम आिण िव�ासासाठी समिप�त. v TABLE OF CONTENTS ABSTRACT……………………………………………………………………………... ii LIST OF TABLES……………………………………………………………………... vii LIST OF FIGURES…………………………………………………………………… viii LIST OF SYMBOLS AND ABBREVIATIONS……………………………………… ix CHAPTER 1. Introduction…………………………………………………………… 1 CHAPTER 2. Mechanisms to generate aneuploidy………………………………….4 CHAPTER 3. Role of p53 in regulating aneuploid cell fate…………………………7 CHAPTER 4. Genome wide screen reveals novel aneuploidy regulators…………12 SUMMARY and DISCUSSION………………………………………………………. 16 APPENDIX A. Methods for Chapter 3 and 4………………………………………... 35 REFERENCES………………………………………………………………………….41 CV………………………………………………………………………………………. 56 vi LIST OF TABLES Table 1 – KEY RESOURCE TABLE 32 vii LIST OF FIGURES Figure 2.1 – Mechanisms to generate aneuploidy and downstream cell fate. 5 Figure 3.1 – Induction of aneuploidy with MPS1i in mammalian cell lines 21 and 3D organotypic cultures. Figure 3.2 – Adherent RPE1 and HCT116 but not Nalm6 cells depend on 22 p53 for growth arrest post aneuploidy induction. Figure 3.3 – 3D organotypic cultures do not activate p53 or undergo 23 growth arrest in response to aneuploidy. Figure 3.4 – TP53-/- mCO exhibit frequent mitotic aberrations 24 Figure 4.1 – Genome wide CRISPR/Cas9 screen reveals novel aneuploidy 25 regulators – Supplementary Figures 26 viii LIST OF SYMBOLS AND ABBREVIATIONS P53 Tumor Suppressor Protein p53 CIN Chromosomal Instability MPS1i Monopolar Spindle Kinase inhibitor mCO Mouse Colon Organoids hMO Human Mammary Organoids EdU 5-Ethynyl-2´-deoxyuridine MPS1i Chromosomal Instability ix CHAPTER 1. INTRODUCTION Aneuploidy refers to the state of unequal chromosome copy numbers and is one of the most prominent genomic aberrations in a majority of solid tumors (Lengauer, Kinzler and Vogelstein, 1998; Weaver and Cleveland, 2006; Beroukhim et al., 2010; Zack et al., 2013; Taylor et al., 2018). In unicellular eukaryotes, it was shown that aneuploidy, by altering the stoichiometry of a large number of genes, can result in dramatic changes in cellular phenotypes and physiology and confer evolutionary adaptation under selective pressure (Selmecki, Forche and Berman, 2006; Torres et al., 2007; Pavelka et al., 2010; Sterkers et al., 2012; Yona et al., 2012; Dephoure et al., 2014; Kaya et al., 2015; Sunshine et al., 2015). Such basic insight about aneuploidy helps explain recent findings that karyotype alterations are associated with cancer initiation as well as the emergence of drug resistance (Lee et al., 2011; Navin et al., 2011; Davoli et al., 2013; Lane et al., 2014; Cai et al., 2016; Graham et al., 2017; Sack et al., 2018; Stichel et al., 2018; Yang et al., 2019). Indeed, cancer may be viewed as a disease of cellular evolution in a multicellular setting, whereby cells of metazoans turn to resembling unicellular organisms that are free to undergo evolutionary adaptation for better survival and proliferation through gross genomic alterations (Nowell, 1976; Duesberg, Stindl and Hehlmann, 2001; Chen et al., 2015; Gerstung et al., 2020). What might differentiate a genomically stable animal organism from the free- adapting unicellular eukaryotes is the presence of p53, acting as the guardian of genome stability by playing key regulatory roles in such processes as the DNA damage response, 1 senescence, and apoptosis (Aylon and Oren, 2011; Reinhardt and Schumacher, 2014; Kastenhuber and Lowe, 2017; Mello and Attardi, 2018; Hafner et al., 2019; Mijit et al., 2020). The loss of functional p53 has been associated with the onset of many metastatic cancers with heightened genomic instability especially on the chromosomal level, whereas an increased p53 gene copy number is thought to be chemopreventive (Sulak et al., 2016; Wasylishen and Lozano, 2016; Bykov et al., 2018; Donehower et al., 2019). Studies in recent years have further suggested roles for p53 in limiting the proliferation of aneuploid cells, however, these studies were limited towards established human cell lines that were chromosomally stable and near diploid, such as RPE1, an hTERT-immortalized retinal pigmented epithelial cell line, HCT116, a colon carcinoma cell line along with a few other cell lines (Cianchi et al., 1999; Li et al., 2010; Thompson and Compton, 2010; Janssen et al., 2011; Kurinna et al., 2013; Hinchcliffe et al., 2016; Potapova et al., 2016; Santaguida et al., 2017; Soto et al., 2017; Giam et al., 2019). Recent studies also revealed complex interplays between p53 and several other genome-protective proteins, such as p38, H3.3 and BCL9L (Hinchcliffe et al., 2016; López-García et al., 2017; Simões-Sousa et al., 2018). However, it has been unclear whether a universal signal elicited by abnormal karyotypes may be sensed by the p53 regulatory mechanism, or karyotype-specific stress states are sensed through diverse mechanisms and converge upon p53 activation. It was also unknown whether cell type or growth environment could contribute to the p53- mediated response to aneuploidy. In my PhD studies, I investigated p53 regulation and downstream cell fate after aneuploidy induction in diverse cell culture models. We set out to answer four questions: 1) How universal is the relation between aneuploidy induction and p53 activation? 2) What are the 2 downstream cell fate consequences after acute aneuploidy induction in different types of cell culture models? 3) Does p53 play a direct role in faithful mitosis rather than sensing aneuploid cells after erroneous mitosis? 4) Identify novel aneuploidy regulators using genome wide CRISR/Cas9 knockout screens? While we confirm that, upon acute aneuploidy induction by treating cells with an inhibitor of the spindle assembly checkpoint (SAC) kinase MPS1 (MPS1i), p53 and p21 were upregulated and cause growth arrest in RPE1 and HCT116 cell lines, this response was not conserved in 3D organotypic cultures of primary cells from mouse and human tissues. Live imaging suggests